Faculty of Science Master Earth Sciences
Research Proposal:
Biodegradability of a novel polymer derived from
structural extracellular polymeric substances
extracted from biofilms in aerobic granular
sludge
Student: Debora Sgarioni Santos Student ID: 10855629 Track: Geo-Ecological Dynamics Examiner: dr. Albert Tietema – IBED/UvA
Assessor: dr. John Parsons – IBED/UvA Daily Supervisor: Yue Wang, MSc – HIMS/UvA
Abstract
Controlled release fertilizers are a better alternative than conventional fertilizers, because they possess a barrier that allows gradual release of nutrients into the soil. However, these barriers are usually non-biodegradable, leading to the accumulation of microplastics in the soil. A new EU Regulation establishes that by 2026 only biodegradable polymers will be allowed in fertilizing products. Recently, structural extracellular polymeric substances (EPS) were extracted from biofilms obtained from aerobic granular sludge and have the trade name Kaumera®. Kaumera has several applications, including as a coating for fertilizers. The aim of this research is to verify if Kaumera is biodegradable according to the EU criteria. Biodegradation rates will be evaluated using three methods: a) respirometry followed by fumigation-extraction, b) weight loss, and c) gas chromatography. The presence of additives on biodegradation rates will be analyzed by adding glycerol in the experiments. Moreover, the influence of soil moisture will be assessed by manipulating this parameter.
Table of Contents
1 INTRODUCTION ... 4
2 THEORETICAL FRAMEWORK... 6
2.1 BIOFILMS ...6
2.2 EXTRACELLULAR POLYMERIC SUBSTANCES ...6
2.3 KAUMERA ...7
2.4 CONTROLLED RELEASE FERTILIZERS ...8
2.5 BIODEGRADATION ... 10
2.6 BIODEGRADABLE POLYMER-COATED FERTILIZERS ... 11
2.7 BIODEGRADABILITY OF EPS ... 12
2.8 EUROPEAN UNION REGULATION ... 13
3 AIM AND RESEARCH QUESTIONS ... 14
4 METHODOLOGY ... 15
4.1 MATERIALS AND EQUIPMENT ... 15
4.2 SOIL MOISTURE ... 15 4.3 RESPIROMETRY ... 16 4.4 MICROBIAL BIOMASS ... 17 4.5 WEIGHT LOSS... 18 4.6 GAS CHROMATOGRAPHY ... 18 4.7 DATA ANALYSIS ... 19 5 EXPECTED RESULTS... 20 6 TIME SCHEDULE ... 21 7 FUNDING ... 21 8 DATA MANAGEMENT ... 22 9 REFERENCES ... 23
1 Introduction
Food demand is projected to increase as the global population reaches 9.7 billion people by 2050 (DESA, 2015). The demand for primary plant nutrients - nitrogen, phosphorus and potassium - in the form of fertilizers increases each year (FAO, 2019). Furthermore, most of the growth in crop production is predicted to originate from higher crop yields and increased cropping intensity. However, the use of fertilizers leads to environmental problems such as water eutrophication, groundwater contamination, soil degradation and air pollution (Chen et al., 2018). These issues are a result of high frequency of fertilizer application and high dosages, which leads to nutrient losses to the environment. Excessive use of fertilizers affects for instance pH and salinity of soils (Lubkowski & Grzmil, 2007).
In order to improve fertilizer efficiency, controlled (CRF) or slow release fertilizers were developed. Their use is not widespread yet due to high costs, thus the sectors that utilize these types of fertilizer are landscaping, horticulture and turf (Chen et al., 2018). The main characteristic of CRFs is that they have some type of barrier that prevents the immediate release of nutrients into the soil and has the aim of matching the nutrient uptake by plants. This barrier is usually in the shape of a coating or a matrix, and it is made of synthetic materials. As a result, after the nutrients are released from CRFs, the coating remains in the soil, which can lead to the accumulation of synthetic polymers and deterioration of soil quality.
A possible solution for the issue of microplastics accumulation in the soil is the use of fertilizer coatings obtained from natural materials that are biodegradable. A few examples are natural polymers obtained from starch, chitosan, lignin, cellulose and biochar (Chen et al., 2018). However, these natural polymers are usually mixed with synthetic polymers to be applied in coatings, since their intrinsic properties are not appropriate as barriers. These blends are considered biodegradable, however, definitions of biodegradability may vary depending on the environmental conditions under which tests are performed (Majeed et al., 2015).
In Europe, the agricultural sector accounts for 5% of plastic waste generated per year (Plastics Europe, 2016). Thus, as part of the European Strategy for Plastics in a Circular Economy, the EU Regulation 2019/1009 on fertilizing products entered into force in 2019. It foresees a restriction in microplastics used in agricultural products by 2021, allowing for a transition period of 5 years for biodegradability criteria to be developed. In 2026, only biodegradable polymers will be allowed in the fertilizer market (European Union, 2019).
Natural polymers can be extracted from biofilms, which are present in the sludge of wastewater treatment stations. Biofilms are aggregates of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS). A new wastewater treatment process, called Nereda®, uses aerobic granular sludge to treat the wastewater. Aerobic granules have a spherical shape and don’t use carrier materials. Nereda is more sustainable compared to other processes due to its smaller footprint, which includes producing less waste.
Recently, EPS were extracted from biofilms obtained from aerobic granular sludge having several industrial applications. The structural fraction of EPS derived from aerobic granular sludge is branded with the trade name Kaumera® (Waterschap Rijn en IJssel, 2019). The combination of Kaumera and other raw materials change the properties of the substance, allowing its use in several applications, such as in agriculture, horticulture and the concrete industry (Waterschap Rijn en IJssel, 2019). The company ChainCraft is currently in the process of testing Kaumera as a coating of controlled release fertilizers.
The use of Kaumera as a building block in different industry sectors contributes to a circular economy, since Kaumera is a waste product and there is a large pool available. Kaumera is a bio-based product, thus the use of materials from fossil fuel sources is avoided. Furthermore, extracting Kaumera from sludge decreases 20-35% of the remaining sludge that needs to be removed and destroyed, decreasing waste. If Kaumera shows to be biodegradable in soil, its application as a coating for fertilizers will avoid the accumulation of microplastics in soil. Hence, the use of biodegradable coatings of natural sources for coated fertilizers contributes to a circular economy, avoiding the use of fossil-fuel based materials and the accumulation of microplastics in the soil. Additionally, the development and research of new types of biodegradable coatings should be incentivized to prevent any type of environmental degradation. Thus, the aim of this research is to analyze the biodegradability of the coating Kaumera in soil, in order to verify if Kaumera complies with the EU Regulation. An assessment of the influence of soil moisture and amount of plasticizers on biodegradation rates will be made.
2 Theoretical Framework
2.1 Biofilms
Biofilms are defined as "aggregates of microorganisms in which cells are frequently embedded in a self-produced matrix of extracellular polymeric substances (EPS) that are adherent to each other and/or a surface" (Vert et al., 2012). Biofilms are widely distributed and are important in biogeochemical processes in all types of environments (Flemming et al., 2016). Functions of the EPS include retention of water, protective barrier, sorption of inorganic and organic compounds, enzymatic activity and nutrient source (Flemming & Wingender, 2010). Biofilms present emergent properties, which are properties that are not anticipated from the study of free-living bacteria. These characteristics include tolerance to desiccation and an external digestive system, among others (Figure 1) (Flemming et al., 2016).
Figure 1. Emergent properties of biofilms and habitat formation (Flemming et al., 2016).
2.2 Extracellular polymeric substances
EPS are considered to be mainly the high-molecular-weight secretions from microorganisms, the outputs of cellular lysis and hydrolysis of macromolecules, together with organic matter adsorbed into the matrix (Sheng et al., 2010). EPS can be divided in bound EPS and soluble EPS. Bound EPS are tightly bound with cells and consist of polymers, sheaths and condensed gels. Soluble EPS are loosely attached to cells or dissolved, and are soluble macromolecules and colloids (Laspidou & Ritmann, 2002). These two types can be separated by centrifugation, with the first forming microbial pellets and the latter remaining in the supernatant. Research on soluble EPS is limited, and most literature refers to bound EPS (Sheng et al., 2010). The composition of EPS is poorly understood, especially regarding molecular composition and function of individual components. This occurs due to their compositional complexity and current challenges in processing and isolating EPS components (Seviour et al., 2019).
Nonetheless, it is clear that the EPS contain a complex mixture of biological polymers, especially polysaccharides, proteins, nucleic acids, lipids and other biopolymers. Their fractions in EPS vary depending on the extraction methods and the sludge sources (Sheng et al 2010). This variation is due to differences in microorganism communities, temperature, availability of nutrients and shear forces (Flemming & Wingender, 2010). Spatial distribution of EPS is described as heterogeneous, and it depends on microbial aggregate types, arrangements and sources. For anaerobic granular sludge, EPS was mostly found in the outer layer, while for aerobic granular sludge the inner layer contained four times more EPS than the
exterior (Wang et al., 2005). The components distribution in EPS is also heterogeneous. It was observed that in aerobic granular sludge the outer layer contained cells and carbohydrates, whereas the inner layer contained most of the proteins (McSwain et al., 2005). There are many functional groups in EPS, like carboxyl, phosphoric, sulfhydryl, phenolic and hydroxyl groups. Due to the presence of hydroxyl and carboxyl groups, EPS have a high binding capacity (Guibaud et al., 2003). EPS molecules contain hydrophilic and hydrophobic groups, indicating the amphoterism feature, which is the capacity of reacting both as an acid and a base (Sheng et al., 2010).
2.3 Kaumera
Biofilms are important in the implementation of wastewater treatment technologies, as well as other industrial applications. EPS provide the structure necessary for anaerobic and aerobic granular sludges, activated sludge and fixed biofilm systems (Seviour et al., 2019). A new wastewater treatment process, called Nereda®, uses aerobic granular sludge to treat the wastewater. Aerobic granule is a type of biofilm of spherical shape without the addition of carrier material. Nereda was developed by Delft University of Technology in a partnership between the University, the Dutch Foundation for Applied Water Research (STOWA), the Dutch Water Authorities and Royal HaskoningDHV (Royal HaskoningDHV, 2020). The advantages of Nereda include lower energy costs, smaller installations, faster settling time (Figure 2), minimal use of chemicals and higher nutrient removal.
Figure 2. Nereda granules (right) settles faster than activated sludge flocs (left). (Retrieved from https://kaumera.com/english/nereda/).
Structural EPS can be extracted from Nereda granules and are called Kaumera Nereda® Gum (Waterschap Rijn en IJssel, 2019). Kaumera is a versatile, bio-based material and can be used as a building block, since when combined with other materials its properties change. The Nereda process is more sustainable than other processes, but still generates waste. The extraction of Kaumera from Nereda sludge granules decreases 20-35% of the waste. Therefore, the use of Kaumera supports the shift towards a circular economy, since it promotes the reuse of a waste product. The company ChainCraft is currently in the process of testing Kaumera as a coating of controlled release fertilizers.
Structural EPS consists of polymers which are able to form hydrogels and contribute to the formation of the network structure of the total EPS (Felz et al., 2016). Hydrogels can take up large amounts of water and swell while preserving its structure. There are several classes of hydrogels, such as ionic gels, temperature-induced gels and pH induced gels. It is likely that the dominant type of hydrogel in the structural EPS are ionic gels, due to the large fraction of
it that was extracted in a study by Felz et al. (2016). The gelling mechanism of structural EPS relates to both the metal ion complexation of the polymers and to the interactions among the present functional groups. (Felz et al., 2020a). Gel stiffness can be analysed by comparing metal preference. The stiffness of structural EPS was higher for transition metals with increasing atomic number, except for copper (Felz et al., 2020a).
Colorimetric methods were used to characterize structural EPS (Felz et al., 2019). Standard compounds were qualitatively assessed, and the overall composition found is the following: amino acids, saccharides, uronic acids, and phenolic compounds. However, quantification of these compounds varies depending on the standard compound used in the methods. Therefore, the authors (Felz et al., 2019) suggest care when interpreting quantitative results. Felz et al. (2020b) studied the presence of glycosaminoglycans in aerobic granular sludge, they were found in the EPS and between cells. The authors speculate that their function is similar to those present in vertebrates: cations attraction, hydrogel forming, protein binding and biological signaling.
2.4 Controlled release fertilizers
Controlled and slow release fertilizers are used to increase crop yield and reduce nutrient losses. However, due to its high cost, their use is still limited to landscaping, horticulture and turf (Chen et al., 2018). In horticulture, environmental conditions can limit nutrient uptake, thus these types of fertilizers might be a solution. Controlled release fertilizers are fertilizers in which the factors responsible for the rate, pattern and duration of release are known and manageable during the CRF manufacture. Slow release fertilizers (SRF), on the other hand, are released slower than common fertilizers but no aspect of release can be regulated. Soil conditions, such as water content, temperature changes and biological activity, as well as handling activities such as storage and transport might affect the release of SRFs (Shaviv, 2001).
Classification of controlled release fertilizers varies according to the literature (Shaviv, 2001; Trenkel, 2010; Lieu et al., 2008; Rose et al., 2002). Azeem et al. (2014) did a comprehensive classification based on the work of those authors (Figure 3), in which the main groups are: 1. Organic compounds, which can be made of natural organic or synthetic organic compounds. The latter can be divided in chemically or biologically degradable.
2. Water soluble fertilizers with physical barriers. These can be divided in coated granules or matrices. Coated granules are made of organic polymer coatings or inorganic coatings. The matrix material can be hydrophobic or hydrophilic. A hydrophilic matrix consists of gel forming polymers – called hydrogels – that are known by their swelling property.
3. Inorganic compounds with low solubility.
Fan & Singh (1990) proposed a different classification, based on method of release control: diffusion, erosion or decomposition, swelling, and osmosis.
Figure 3. Classification of controlled release fertilizers, the main groups are: organic compounds, water soluble fertilizers with physical barriers, and inorganic compounds with low solubility (Azeem et al., 2014).
The controlled release mechanism depends on several factors such as type of CRF, origin of coating, and environmental conditions. The widespread application of CRFs is limited by a lack of data concerning release mechanisms in diverse soil conditions (Azeem et al., 2014). Nonetheless, Shaviv (2001) proposed a multi-stage release mechanism for coated fertilizers. The first stage is the penetration of water, mainly water vapor, which condenses and dissolves part of the nutrients in the core, increasing internal pressure. From here the granule swells and there are two possible paths (Figure 4). If the osmotic pressure is higher than the membrane resistance, the coating breaks and the fertilizer is released immediately, this is called failed mechanism or catastrophic release. If the membrane can endure the pressure, the fertilizer is released through diffusion. Diffusion can occur via concentration gradient or pressure gradient, or both.
Figure 4. Scheme of release stages from a polymer-coated granule (Shaviv, 2001).
Advantages of using CRFs include lower application frequency and lower nutrient losses. However, a few issues arise from the use of coated fertilizers. For example, the last stage of
nutrient release might be too slow due to a decreased concentration gradient, resulting in 10-30% of nutrients left (Ge et al., 2002). Furthermore, some coating materials can be toxic or non-biodegradable in the soil (Azeem et al., 2014), contributing to soil contamination.
2.5 Biodegradation
Biodegradation is the breakdown of matter due to the action of enzymes secreted by microorganisms, leading to lower molecular weight products (Vroman & Tighzert, 2009). Moreover, biodegradation of polymeric materials includes several steps: biodeterioration, depolymerization/biofragmentation, assimilation, and mineralization (Lucas et al., 2008). Biodegradation of polymers is affected by environmental conditions and properties of the material (Majeed et al., 2015).
Biodeterioration is the first step, in which microorganisms, other decomposers and abiotic factors break the material into small particles. The process of polymer breakdown is called depolymerization, in which microorganisms secrete catalytic agents (enzymes and free radicals) that reduce the molecular weight of polymers (Lucas et al., 2008). Enzymes are proteins with complex chemical structures that possess hydrophilic groups and cleave the molecular chains into segments, generating oligomers, dimers and monomers (Mohan, 2011). Low molecular weight compounds are more accessible to chemical reactions. These enzymes are divided into two groups, extracellular depolymerase and intracellular depolymerase (Gu, 2003).
Assimilation occurs in the cytoplasm, where small molecules are incorporated into the microbial metabolism and are used to produce biomass, energy, and several primary and secondary metabolites (Lucas et al., 2008). In microbial metabolism, the breakdown of complex molecules into simpler molecules in order to be assimilated releases energy and is called catabolism, whereas anabolism builds complex molecules, forming new microbial biomass (Chiellini et al., 2007). Mineralization is the last step but it is not necessarily reached. The end products are inorganics compounds, like CO2, H2O, or CH4 and secreted metabolites
(organic acids, aldehydes, terpenes) (Mohan, 2011). There are three catabolic pathways for microorganisms: aerobic respiration, anaerobic respiration, and fermentation, which are followed depending on the presence and type of electron transport systems (Lucas et al., 2008). The rate of biodegradation varies depending on the functional groups and degree of complexity of polymers (Karlsson & Albertsson, 1998). Structural elements such as branching of chains or the formation of networks (cross linked polymers) can influence accessibility of the material to polymer chain cleavage (Mohan, 2011). Other factors that influence biodegradation are polymer origin, hydrophobic/hydrophilic characteristics, and the presence of additives, such as plasticizers. Plasticizers are used to increase the flexibility of polymers, which occurs via a reduction in intermolecular forces along polymer chains, increasing chain mobility. The second most used plasticizer is glycerol (Vieira et al., 2010). Glycerol may facilitate biodegradation of biopolymers in soil by increasing water vapor permeability, turning them more hydrophilic and more accessible to microorganisms (Suyatma et al., 2005). That was observed by Dean et al. (2013), they compared biodegradation rates of different formulations of chitosan films in soil. Glycerol-plasticized chitosan achieved higher biodegradation rates than pure chitosan. Soil properties may influence biodegradation rates of different polymers. Hoshino et al (2001) found that temperature and total nitrogen in the soil affected the biodegradation of several polymers. Hamarashid et al. (2010) showed that soil texture also plays a role in biodegradation, with biodegradation rates of plants residues being higher in fine soils than in coarser soils.
Amount of organic matter and total nitrogen were also correlated with biodegradation. Another study (Yamamoto-Tamura et al., 2015) found that the varying biodegradation rates of a polymer in a range of soil textures was associated with the distribution of a type of fungi. Grima et al. (2000) considered that among environmental factors affecting aerobic biodegradation of polymers in soils, namely temperature, moisture, salts and oxygen, the most significant factor is moisture. Biodegradation of gelatin-based films in soil was studied by Martucci & Ruseckaite (2009). They observed that microbial attack depended on the moisture absorption capacity of materials present in the different formulations.
2.6 Biodegradable Polymer-coated Fertilizers
Biodegradable polymer-coated controlled release fertilizers are a possible solution to achieve high crop yields, reduce nutrient losses, and avoid environmental contamination of the soil, specially by microplastics (Majeed et al., 2015). The perception of biodegradability may vary depending on the purpose of use and the environmental conditions under which tests are performed (Majeed et al., 2015), hence some polymer blends are considered biodegradable due to the addition of natural polymers. A few examples of such polymers are starch, chitosan, lignin and cellulose (Chen et al., 2018). However, natural polymers are often used in blends with synthetic polymers in coated fertilizers, because in their pure form they are not effective as an encapsulating material. Examples of blends used as coated fertilizers are chitosan-clay composite (dos Santos et al., 2015), modified starch with polylactic acid (Chen et al., 2008), carboxymethyl cellulose matrix (Davidson et al., 2013), lignin-polyurethane composite (Peng & Chen, 2011).
As mentioned in section 2.4, hydrogels can be the components of coatings, especially in biodegradable coatings. Hydrogels are able to increase their size via water absorption and release their content gradually (Zhong et al., 2013). As can be seen in Figure 5, hydrogel swelling allows the slow nutrient release, then biodegradation starts via disintegration of the matrix, followed by biofragmentation caused by enzymatic activity of microorganisms. Depolymerization results in lower molecular compounds that are assimilated by microbes and are mineralized into final products such as CO2 or CH4 and microbial biomass. Advantages of
using hydrogel forming biodegradable coatings include improvement of the water holding capacity of the soil and particle aggregation, while reducing oxidative stress (Burke et al., 2010).
Figure 5. Scheme of nutrient release and biodegradation processes of hydrogel forming biodegradable polymer-coated fertilizer in soil (Majeed et al., 2015).
2.7 Biodegradability of EPS
Extraction method can determine the biodegradability of EPS. Park & Novak (2007) reported that the EPS from activated sludge flocs showed different biodegradability depending on the extraction methods. Certain methods favored anaerobic degradation while others favored aerobic. Physical extraction methods were found to extract mainly hydrophilic components from activated sludge (Wei et al., 2012), and those components were readily biodegraded. Hence, biodegradation rates of EPS are also correlated with the hydrophobic/hydrophilic characteristics of the EPS.
Wang et al. (2005) submitted aerobic granules to a starvation test, and observed that the core of the granule became hollow, while the outer layer was intact. The authors also analysed hydrophobicity in aerobic granules, and they found a higher hydrophobicity in the outer layer of the granule than in the core. Furthermore, EPS content was determined and five times more EPS was found in the core compared to the outer layer. They concluded that the outer layer is made of poorly soluble and non-easily biodegradable EPS, while the core consists of soluble and readily biodegradable EPS. Hsieh et al. (1994) suggested that only soluble EPS are biodegradable, and that soluble EPS are released from bound EPS and are consumed by active cells. Wang et al. (2007) extracted EPS from aerobic granules and observed similar results as Wang et al. (2005). Part of the EPS from aerobic granule was biodegraded by their own producers, and the non-readily biodegradable part is believed to play an essential role in the structural integrity of the granule.
Zhang & Bishop (2003) investigated if EPS can be degraded by microorganisms other than their own producers. EPS were extracted from biofilm reactors (rotating drum) and compared with EPS from activated sludge. They found that EPS can be used as a substrate, and carbohydrates were consumed faster than proteins according to colorimetric methods.
2.8 European Union Regulation
As part of the European Strategy for Plastics in a Circular Economy, the EU Regulation 2019/1009 on fertilizing products entered into force in 2019. It predicts a restriction in microplastics used in agricultural products by 2021, allowing for a transition period of 5 years for biodegradability criteria to be developed. In 2026, only biodegradable polymers will be allowed in the fertilizer market (European Union, 2019).
By 2024, the European Commission will assess biodegradability criteria for polymers and test methods to check compliance with those criteria. The criteria should ensure that:
(a) the polymer is capable of undergoing physical and biological decomposition in natural soil conditions and aquatic environments across the Union, so that it ultimately decomposes only into carbon dioxide, biomass and water;
(b) the polymer has at least 90 % of the organic carbon converted into carbon dioxide in a maximum period of 48 months after the end of the claimed functionality period of the EU fertilizing product indicated on the label, and as compared to an appropriate standard in the biodegradation test; and
3 Aim and Research Questions
The aim of this research is to analyze the biodegradability of Kaumera in soil, in order to verify if Kaumera complies with the EU Regulation and can be considered a biodegradable material. Moreover, an assessment of the influence of soil moisture and amount of added plasticizers on biodegradation rates will be conducted.
Main research question is the following:
• Are the Kaumera formulations biodegradable according to the EU criteria? Sub-questions are:
• How does soil moisture affect the biodegradability rate of Kaumera?
4 Methodology
To analyse the biodegradability of different formulations of Kaumera, the biodegradation rate in soils will be evaluated using three methods: a) respirometry followed by fumigation-extraction, b) weight loss, and c) gas chromatography. To analyse how soil moisture affects biodegradation rates, we will manipulate this property in the weight loss and gas chromatography experiments. The inhibitory effect of glycerol as a plasticizer in biodegradation processes will be assessed by adding this substance in all the experiments.
4.1 Materials and Equipment
Eight different types of Kaumera formulations, with different types and quantities of plasticizers, will be made available by ChainCraft. Quantities of the material can be seen in Table 2. Kaumera will be used in the form of sheets. The soil samples used as substrate will be a commercially available and well described standard soil (Table 1).
Table 1. Characteristics of the standard soil.
Standard soil type nr 2.3
Organic carbon (%C) 0.65
Nitrogen (%N) 0.07
pH value 6.1
CEC (meq/100g) 6.8
Soil texture sandy loam
Maximum water holding
capacity (g/100g) 35.2
Table 2. Quantities of material used in each experiment.
Formulations of Kaumera Experiments 1 2 3 4 6 7 8 Respicond 1.8 1.8 1.8 1.8 1.8 1.8 1.8 GC 4.5 4.5 4.5 - - - - Weight Loss 13.5 13.5 13.5 - - - - Total (g) 19.8 19.8 19.8 1.8 1.8 1.8 1.8
The equipments used will be the Respicond, for measurements of CO2 production, the Elemental Analyser for measurements of C, N and P, Gas Chromatograph for analysis of CH4
production, the Auto Analyser, an incubator, an oven, a desiccator, a fridge, scales and flasks.
4.2 Soil moisture
The influence of soil moisture on biodegradation rates of three Kaumera formulations will be assessed with a manipulation experiment, in which water will be added to dried soils.
Percentages of soil moisture will be defined to represent three categories: dry, moist, and extremely wet. This property will be analysed in the weight loss and gas chromatography experiments.
4.3 Respirometry
Respirometry is a method that measures the amount of carbon transformed into CO2 by the activity of microorganisms. The experiment will be conducted according to the ISO 17556:2019, and the duration will be of 60 days.
1. Preparation of materials
Eight formulations of Kaumera will be tested. The TOC (total organic carbon) will be determined by elemental analysis, then ThCO2 (theoretical amount of carbon dioxide evolved by the test material) can be calculated in the end of the experiment. 300mg of Kaumera will be used with 15g of dried soil. As a reference material, a well-defined biodegradable polymer (cellulose) will be used. The physical form and shape of all materials should be the same. 2. Preparation of soil
The soil will be sieved in a 2 mm sieve, and the following properties will be measured: soil moisture, pH and organic matter content. The soil will be stored in a sealed container at 4 °C until further use.
3. Execution of the experiment
The outlook of the experiment can be seen below (Table 3). The flasks will be placed in the Respicond, at 25°C and CO2 will be measured at a determined interval.
Table 3. Respirometry experiment.
Respicond Experiment
Standard soil 1
Water content constant 1
Formulations (8, glycerol, cellulose and blank) 11
Replicates 6
Nr of samples 66
Empty flasks 4
Total nr of flasks 70
4. Calculations
The ThCO2 (theoretical amount of carbon dioxide evolved by the test material) is given in milligrams by:
𝑇ℎ𝐶𝑂2 =44
where: 𝑚 is the mass of the test material in mg, 𝑤𝑐 is the carbon content of the test material expressed as a mass fraction, 44 is the molecular mass of carbon dioxide, and 12 is the atomic mass of carbon.
The percentage biodegradation Dt is calculated for each test flask for each measurement interval as:
𝐷𝑡 =
∑ 𝑚𝑇− ∑ 𝑚𝐵 𝑇ℎ𝐶𝑂2
× 100
∑ 𝑚𝑇 is the amount of carbon dioxide evolved in the test flask between the start of the test and time t
∑ 𝑚𝐵 is the amount of carbon dioxide evolved in the blank flask between the start of the test and time t
ThCO2 and Dt will be also calculated for the reference material (cellulose).
Furthermore, the final carbon content 𝐶𝑓𝑖𝑛𝑎𝑙 remaining in the test material can be obtained as: 𝐶𝑓𝑖𝑛𝑎𝑙 =12
44× (∑ 𝑚𝑇− ∑ 𝑚𝐵)
And the percentage of carbon 𝐶𝑏 that was biodegraded is calculated as: 𝐶𝑏= 𝐶𝑓𝑖𝑛𝑎𝑙
𝑚 × 𝑤𝑐 × 100
4.4 Microbial Biomass
Besides the production of CO2, in aerobic conditions the heterotrophic microbial community also utilizes the carbon source for production of new biomass. The goal of this experiment is to determine the polymer-derived carbon incorporated into microbial biomass after the incubation time.
Microbial biomass can be measured by Fumigation Extraction (FE) (Vance et al., 1987), in which the fumigant (chloroform) causes cell lysis of microorganisms, and releases a potassium sulfate (K2SO4) solution from which carbon can be extracted. The soil in each flask from the
respirometer experiment will be divided in 2 samples, one will be fumigated and the other won’t. Carbon concentrations in extraction solution of unfumigated and fumigated soil are analyzed by an infrared analyzer of total organic carbon (TOC). The following equation is applied:
𝐶 = 𝑘𝐸𝐶 × (𝐸𝐹− 𝐸𝑁)
where 𝐶 is the carbon incorporated into the microbial biomass, 𝐸𝐹 is the organic carbon extracted from fumigated soil, 𝐸𝑁 is the organic carbon extracted from non-fumigated soil,
If the carbon content of Kaumera is known previously to the degradation period, it is possible to know the amount of carbon that was lost as CO2 and that was transferred from Kaumera to
the microorganisms in the form of biomass.
4.5 Weight Loss
300mg of Kaumera will be placed with 50g of soil in a flask, which will be incubated at 25°C. The duration of the experiment will be of 60 days, and samples will be collected at a determined interval in 5 times (Table 4). The Kaumera sheets will be cleaned, dried and weighed and the weight loss will be calculated as described below.
Table 4. Weight Loss experiment.
Weight Loss Experiment
Standard soil 1
Water content (dry, moist, extremely wet) 3
Formulations (3 and glycerol) 4
Replicates 3
Sampling times 5
Nr of samples 180
Degradation rates will be indicated by the percentage weight loss (𝑊𝐿) of the samples with
time, as follows:
𝑊𝐿= [(𝑀0− 𝑀1)
𝑀0 ] × 100%
where 𝑀0 is the initial mass of the film before the test, and 𝑀1is the residual mass of the film after the test.
4.6 Gas Chromatography
Gas Chromatography will be used to assess CO2 and CH4 levels, in order to investigate aerobic
and anaerobic respiration in the samples with varying water contents. 300mg of Kaumera will be placed with 50g of soil in airtight serum bottles. The air inside the bottle will be replaced by synthetic air, which is a mix of pure oxygen and pure nitrogen. The pressure inside the bottles will be measured and stabilized. Incubation of samples will occur at 25°C. The bottles will be refreshed with oxygen when necessary. Gas samples will be taken out of the serum bottles and will be placed in smaller bottles for GC measurements. This is done to avoid interruption of biodegradation. The outlook of the experiment can be seen below (Table 5).
Table 5. Gas Chromatography experiment
Gas Chromatography Experiment
Water content (dry, moist, extremely wet) 3
Formulations (3, glycerol and blank) 5
Replicates 3
Nr of samples 45
Calibration of the GC is necessary to obtain retention times of CO2 and CH4 and quantification.
Standard gases with known concentrations of CO2 and CH4 will be used and from them
calibration curves will be made, correlating area and element concentration. Chromatographs will provide the peak area of the elements, and from the calibration curve the concentration (𝐶𝑜𝑛𝐸 ) is found. Further, volume of the element (𝑉𝐸 ),which can be CO2 or CH4, is found as:
𝑉𝐸 = 𝑉𝐻𝑆 × 𝐶𝑜𝑛𝐸
Volume of the headspace of the bottle ( 𝑉𝐻𝑆 ) is obtained by adding water to a bottle, weighing and dividing by the water density. The ideal gas law provides the total amount of gas ( 𝑛𝑇 ) in the bottle:
𝑛𝑇 =
(𝑃 × 𝑉𝐻𝑆) 𝑅 × 𝑇
P is the pressure measured from the bottle, R is the ideal gas constant, and T is the temperature. The same law can be used for the volume of CO2 in the headspace of the bottle, thus the amount
of CO2 (𝑛𝐶𝑂2 ) can be found by using the ratio of volumes and the total amount of gas (nT).
𝑛𝐶𝑂2 = 𝑛𝑇 × (𝑉𝐶𝑂2 𝑉𝐻𝑆)
The same is done for methane:
𝑛𝐶𝐻4 = 𝑛𝑇 × (𝑉𝐶𝐻4 𝑉𝐻𝑆) Finally, the percentage of biodegradation (Dt) is calculated:
𝐷𝑡 = ∑ 𝑚𝑇− ∑ 𝑚𝐵
𝑇ℎ𝐶𝑂2 × 100
4.7 Data Analysis
Concerning data analysis, statistically significant differences will be determined using ANOVA test in R software.
5 Expected Results
The expected results of this research are the following:
• It is unknown if Kaumera formulations are biodegradable in soil. The biodegradation of structural EPS has been analysed only in aqueous solution until now, and parts of EPS were found to be readily biodegradable, whereas the non-readily biodegradable part was suggested to be responsible for the structure of the granule.
• Concerning soil moisture, it is expected that an optimum level of water content accelerates aerobic biodegradation in soil. An extremely wet soil may affect the access to oxygen by microbial biota, which may promote anaerobic respiration.
• The presence of glycerol as a plasticizer in Kaumera may increase biodegradation rates. Since glycerol may increase the hydrophilicity of Kaumera, the access of soil microbiota to this material would be improved.
6 Time Schedule
The preliminary time schedule of the research is as follows (Figure 6). A summer break (B) will occur for one week in July.
Figure 6. Preliminary time schedule of the research.
7 Funding
The research costs are estimated in the table below (Table 6). The laboratory equipment is already owned by the University. Regarding the materials, soil, cellulose and glycerol will be provided by the lab from HIMS, and Kaumera samples will be provided by ChainCraft.
Table 6. Estimated research costs
Research Costs Quantity Price (€)
Lab Equipment Respicond 1 65500 Elemental Analyser 1 50000 Auto Analyser 1 80000 Gas Chromatograph 1 60000 Oven 1 1000 pH meter 1 400 Desiccator 1 200 Incubator 1 1000 Sieve 1 20
Wide neck bottles 180 100
Serum bottles 45 80 Total (€) 258300 Materials Soil 11kg 88 Cellulose 1g 10 Glycerol 100ml 5 Kaumera 67g - Total (€) 103 Total Costs (€) 258403
8 Data Management
The data obtained with this research will be made publicly available at GitHub, in accordance with the FAIR (Findable, Accessible, Interoperable and Reusable) principle (Wilkinson et al., 2016).
9 References
Azeem, B., KuShaari, K., Man, Z. B., Basit, A., & Thanh, T. H. (2014). Review on materials & methods to produce controlled release coated urea fertilizer. Journal of controlled release, 181, 11-21.
Burke, D. R., Akay, G., & Bilsborrow, P. E. (2010). Development of novel polymeric materials for agroprocess intensification. Journal of applied polymer science, 118(6), 3292-3299.
Chen, J., Lü, S., Zhang, Z., Zhao, X., Li, X., Ning, P., & Liu, M. (2018). Environmentally friendly fertilizers: A review of materials used and their effects on the environment. Science of the total environment, 613, 829-839.
Chen, L., Xie, Z., Zhuang, X., Chen, X., & Jing, X. (2008). Controlled release of urea encapsulated by starch-g-poly (L-lactide). Carbohydrate starch-g-polymers, 72(2), 342-348.
Chiellini, E., Corti, A., D’Antone, S., & Billingham, N. C. (2007). Microbial biomass yield and turnover in soil biodegradation tests: carbon substrate effects. Journal of Polymers and the Environment, 15(3), 169-178. Davidson, D. W., Verma, M. S., & Gu, F. X. (2013). Controlled root targeted delivery of fertilizer using an
ionically crosslinked carboxymethyl cellulose hydrogel matrix. SpringerPlus, 2(1), 1-9.
Dean, K., Sangwan, P., Way, C., Zhang, X., Martino, V. P., Xie, F., ... & Avérous, L. (2013). Glycerol plasticised chitosan: A study of biodegradation via carbon dioxide evolution and nuclear magnetic resonance. Polymer degradation and stability, 98(6), 1236-1246.
DESA, U. (2015). World population prospects: The 2015 revision, key findings and advance tables. United Nations Department of Economic and Social Affairs. Population Division working paper no
ESA/P/WP, 241.
dos Santos, B. R., Bacalhau, F. B., dos Santos Pereira, T., Souza, C. F., & Faez, R. (2015). Chitosan-montmorillonite microspheres: a sustainable fertilizer delivery system. Carbohydrate polymers, 127, 340-346.
European Union (2019). Regulation 2019/1009 of the European Parliament. Official Journal of the European
Union. Retrieved from
https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=OJ:L:2019:170:FULL&from=EN
Fan, L. T., & Singh, S. K. (1990). Controlled release: A quantitative treatment (Vol. 13). Springer Science & Business Media.
FAO (2019). World fertilizer trends and outlook to 2020. Rome: Food and Agriculture Organization of the United Nations. Retrieved from http://www.fao.org/3/a-i6895e.pdf
Felz, S., Al-Zuhairy, S., Aarstad, O. A., van Loosdrecht, M. C., & Lin, Y. M. (2016). Extraction of structural extracellular polymeric substances from aerobic granular sludge. JoVE (Journal of Visualized
Experiments), (115), e54534.
Felz, S., Kleikamp, H., Zlopasa, J., van Loosdrecht, M. C., & Lin, Y. (2020a). Impact of metal ions on structural EPS hydrogels from aerobic granular sludge. Biofilm, 2, 100011.
Felz, S., Neu, T. R., van Loosdrecht, M. C., & Lin, Y. (2020b). Aerobic granular sludge contains Hyaluronic acid-like and sulfated glycosaminoglycans-acid-like polymers. Water research, 169, 115291.
Felz, S., Vermeulen, P., van Loosdrecht, M. C., & Lin, Y. M. (2019). Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS). Water research, 157, 201-208.
Flemming, H. C., & Wingender, J. (2010). The biofilm matrix. Nature reviews microbiology, 8(9), 623.
Flemming, H. C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., & Kjelleberg, S. (2016). Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology, 14(9), 563.
Ge, J., Wu, R., Shi, X., Yu, H., Wang, M., & Li, W. (2002). Biodegradable polyurethane materials from bark and starch. II. Coating material for controlled‐release fertilizer. Journal of applied polymer science, 86(12), 2948-2952.
Grima, S., Bellon-Maurel, V., Feuilloley, P., & Silvestre, F. (2000). Aerobic biodegradation of polymers in solid-state conditions: a review of environmental and physicochemical parameter settings in laboratory simulations. Journal of Polymers and the Environment, 8(4), 183-195.
Gu, J. D. (2003). Microbiological deterioration and degradation of synthetic polymeric materials: recent research advances. International biodeterioration & biodegradation, 52(2), 69-91.
Guibaud, G., Tixier, N., Bouju, A., & Baudu, M. (2003). Relation between extracellular polymers’ composition and its ability to complex Cd, Cu and Pb. Chemosphere, 52(10), 1701-1710.
Hamarashid, N. H., Othman, M. A., & Hussain, M. A. H. (2010). Effects of soil texture on chemical compositions, microbial populations and carbon mineralization in soil. Egypt. J. Exp. Biol.(Bot.), 6(1), 59-64.
Hoshino, A., Sawada, H., Yokota, M., Tsuji, M., Fukuda, K., & Kimura, M. (2001). Influence of weather conditions and soil properties on degradation of biodegradable plastics in soil. Soil science and plant
nutrition, 47(1), 35-43.
Hsieh, K. M., Murgel, G. A., Lion, L. W., & Shuler, M. L. (1994). Interactions of microbial biofilms with toxic trace metals: 1. Observation and modeling of cell growth, attachment, and production of extracellular polymer. Biotechnology and bioengineering, 44(2), 219-231.
Karlsson, S., & Albertsson, A. C. (1998). Biodegradable polymers and environmental interaction. Polymer
Engineering & Science, 38(8), 1251-1253.
Laspidou, C. S., & Rittmann, B. E. (2002). A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water research, 36(11), 2711-2720.
Liu, L., Kost, J., Fishman, M. L., & Hicks, K. B. (2008). A review: controlled release systems for agricultural and food applications. New Delivery Systems for Controlled Drug Release from Naturally Occurring
Materials, ACS Symposium series, 992, pp. 265-281
Lubkowski, K., & Grzmil, B. (2007). Controlled release fertilizers. Polish Journal of Chemical Technology, 9(4), 83-84.
Lucas, N., Bienaime, C., Belloy, C., Queneudec, M., Silvestre, F., & Nava-Saucedo, J. E. (2008). Polymer biodegradation: Mechanisms and estimation techniques–A review. Chemosphere, 73(4), 429-442. Majeed, Z., Ramli, N. K., Mansor, N., & Man, Z. (2015). A comprehensive review on biodegradable polymers
and their blends used in controlled-release fertilizer processes. Reviews in Chemical Engineering, 31(1), 69-95.
Martucci, J. F., & Ruseckaite, R. A. (2009). Biodegradation of three-layer laminate films based on gelatin under indoor soil conditions. Polymer Degradation and Stability, 94(8), 1307-1313.
McSwain, B. S., Irvine, R. L., Hausner, M., & Wilderer, P. A. (2005). Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Appl. Environ. Microbiol., 71(2), 1051-1057.
Mohan, K. (2011). Microbial deterioration and degradation of polymeric materials. Journal of Biochemical
Technology, 2(4), 210-215.
Park, C., & Novak, J. T. (2007). Characterization of activated sludge exocellular polymers using several cation-associated extraction methods. Water research, 41(8), 1679-1688.
Peng, Z., & Chen, F. (2011). Synthesis and properties of lignin-based polyurethane hydrogels. International
Journal of Polymeric Materials, 60(9), 674-683.
Plastics Europe (2016). Plastics–the Facts. An analysis of European plastics production, demand and waste data.
Retrived from: http://www. plasticseurope.org.
Rose, R., Dumroese, R. K., Riley, L. E., & Landis, T. D. (2002). Slow release fertilizers 101. National
Proceedings of the Forest and Conservation Nursery Associations. 1999-2001. RMRS-P-24., 304-308.
Royal HaskoningDHV. (2020). About Nereda Technology. Retrieved from https://www.royalhaskoningdhv.com/en-gb/nereda/about-nereda-technology
Seviour, T., Derlon, N., Dueholm, M. S., Flemming, H. C., Girbal-Neuhauser, E., Horn, H., ... & Nerenberg, R. (2019). Extracellular polymeric substances of biofilms: Suffering from an identity crisis. Water research,
151, 1-7.
Shaviv, A. (2001). Advances in controlled-release fertilizers. Advances in agronomy, 71(1), 1-49.
Sheng, G. P., Yu, H. Q., & Li, X. Y. (2010). Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnology advances, 28(6), 882-894.
Suyatma, N. E., Tighzert, L., Copinet, A., & Coma, V. (2005). Effects of hydrophilic plasticizers on mechanical, thermal, and surface properties of chitosan films. Journal of Agricultural and Food Chemistry, 53(10), 3950-3957.
Trenkel, M. E. (2010). Slow-and controlled-release and stabilized fertilizers: An option for enhancing nutrient
use efficiency in agriculture. IFA, International fertilizer industry association.
Vance, E. D., Brookes, P. C., & Jenkinson, D. S. (1987). An extraction method for measuring soil microbial biomass C. Soil biology and Biochemistry, 19(6), 703-707.
Vert, M. et al. Terminology for biorelated polymers and applications (IUPAC Recommendations 2012). Pure
Vieira, M. G. A., da Silva, M. A., dos Santos, L. O., & Beppu, M. M. (2011). Natural-based plasticizers and biopolymer films: A review. European Polymer Journal, 47(3), 254-263.
Vroman, I., & Tighzert, L. (2009). Biodegradable polymers. Materials, 2(2), 307-344.
Wang, Z. W., Liu, Y., & Tay, J. H. (2005). Distribution of EPS and cell surface hydrophobicity in aerobic granules. Applied microbiology and biotechnology, 69(4), 469.
Wang, Z. W., Liu, Y., & Tay, J. H. (2007). Biodegradability of extracellular polymeric substances produced by aerobic granules. Applied microbiology and biotechnology, 74(2), 462-466.
Waterschap Rijn en IJssel, 2019. Kaumera. Retrieved from https://kaumera.com/
Wei, L. L., Wang, K., Zhao, Q. L., Jiang, J. Q., Kong, X. J., & Lee, D. J. (2012). Fractional, biodegradable and spectral characteristics of extracted and fractionated sludge extracellular polymeric substances. Water
research, 46(14), 4387-4396.
Wilkinson, M. D., Dumontier, M., Aalbersberg, I. J., Appleton, G., Axton, M., Baak, A., ... & Bouwman, J. (2016). The FAIR Guiding Principles for scientific data management and stewardship. Scientific data, 3. Yamamoto-Tamura, K., Hiradate, S., Watanabe, T., Koitabashi, M., Sameshima-Yamashita, Y., Yarimizu, T., &
Kitamoto, H. (2015). Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils. AMB Express, 5(1), 10.
Zhang, X., & Bishop, P. L. (2003). Biodegradability of biofilm extracellular polymeric substances. Chemosphere, 50(1), 63-69.
Zhong, K., Lin, Z. T., Zheng, X. L., Jiang, G. B., Fang, Y. S., Mao, X. Y., & Liao, Z. W. (2013). Starch derivative-based superabsorbent with integration of water-retaining and controlled-release fertilizers. Carbohydrate
